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Title: Role of Siglec-7 in ganglioside recognition and modulating NK cell biology
Author: Mohan, Bindu
Awarding Body: University of Dundee
Current Institution: University of Dundee
Date of Award: 2013
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Sialic acid binding Ig-like lectin-7 (Siglec-7), expressed primarily on NK cells, binds preferentially to alpha2,8 linked disialic acids such as present in the ganglioside GD3 that is upregulated in certain cancers. Siglec-7 is classified as an inhibitory receptor as it contains immunoreceptor tyrosine based inhibitory motifs. It has been shown to inhibit NK cytotoxicity in cellular assays thereby implying a role for it in NK cell mediated tumour surveillance. The aim of this project was to study factors affecting ligand recognition by Siglec-7 and its impact on NK cell functions. An investigation into the mechanism by which Siglec-7 mediates inhibitory signals to regulate NK cell biology was also carried out. Recognition of Siglec-7 for GD3 has been reported to be altered in the presence of complex gangliosides. This project was initiated with an aim to examine the role of such cis-interactions between GD3 and other gangliosides such as GM1 in biological systems and thereby its impact on NK cell biology. B16 (78) cell line was genetically modified to over-express both GD3 and GM1. This model system was then analysed using Siglec-7-Fc precomplexes for the recognition of GD3. Siglec-7-Fc binding of B16 (78) cells with high expression of GD3 and GM1 was significantly lower compared to cells having high expression of GD3 and low expression of GM1. However further investigation of these cis-interactions by confocal microscopy revealed that only less than 3% of the cells had patches of co-localization of the two gangliosides. Such lateral segregation of co-expressed GD3 and GM1 was also observed in another cell line model. Next, an investigation into the role of GD3 in modulating NK cell functions via Siglec-7 was carried out. Primary PBMCs and a Siglec-7 deficient NK cell line, NK92, were used for this purpose. The data obtained showed that Siglec-7 could negatively modulate NK cytotoxicity towards targets expressing disialylated ligands such as GD3. Furthermore Siglec-7 was also able to modulate integrin functions on NK cells. LFA-1 mediated adhesion of effectors to ICAM-1-Fc coated plates and the polarization of perforin granules to ICAM-1- Fc coated beads were negatively affected by the expression of Siglec-7 in the NK92 cell line. Biochemical analysis of LFA-1 mediated signalling in NK92 cells showed negative regulation of Src kinase activation, in an Siglec-7 dependent manner. Overall these findings suggest a role for Siglec-7 in modulating NK cell recognition of tumours with aberrant glycosylation patterns. They also form the basis of further investigation into the mechanisms of inhibitory signalling mediated by Siglec-7 and could therefore be of potential clinical relevance in NK cell mediated tumour clearance.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available